U.S. patent number 7,700,326 [Application Number 11/454,524] was granted by the patent office on 2010-04-20 for rt-pcr detection for differential diagnosis of field isolates or lapinized vaccine strain of classical swine fever virus (csfv) in samples.
This patent grant is currently assigned to Animal Health Research Institute, Council of Agriculture, Executive Yuan. Invention is credited to Ming-Hwa Jong, Chu-Hsiang Pan.
United States Patent |
7,700,326 |
Pan , et al. |
April 20, 2010 |
RT-PCR detection for differential diagnosis of field isolates or
lapinized vaccine strain of classical swine fever virus (CSFV) in
samples
Abstract
The present invention provides a rapid RT-PCR detection method
and a diagnostic kit for differentiating field isolates of
classical swine fever virus (CSFV) from lapinized CSF vaccine
viruses in samples. In order to detecting different genotypes of
CSF virus, this invention uses a pair (or pairs) of CSF virus
specific primers designed from the conserved sequences within the
3'-untranslated region of CSFV which contains an insertion of
12.about.13 nucleotides (poly T) in the region of the lapinized CSF
vaccine virus, in comparison with the corresponding region of field
isolates of CSFV. By the RT-PCR or the RT-PCR followed by a
nest-PCR, field isolates of CSFV and lapinized CSF vaccine viruses
in samples could be detected directly and quickly and/or
differentiated in electrophoresis without further enzymatic
digestion or DNA sequencing.
Inventors: |
Pan; Chu-Hsiang (Taipei County,
TW), Jong; Ming-Hwa (Taipei County, TW) |
Assignee: |
Animal Health Research Institute,
Council of Agriculture, Executive Yuan (Taipei County,
TW)
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Family
ID: |
37573815 |
Appl.
No.: |
11/454,524 |
Filed: |
June 16, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060286551 A1 |
Dec 21, 2006 |
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Foreign Application Priority Data
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Jun 17, 2005 [TW] |
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94120270 A |
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Current U.S.
Class: |
435/91.2 |
Current CPC
Class: |
C12Q
1/701 (20130101) |
Current International
Class: |
C12P
19/34 (20060101) |
Field of
Search: |
;435/91.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
McGoldrick et al. Journal of Virological Methods, vol. 79, pp.
85-95, 1999. cited by examiner .
Chu-Hsiang Pan et al.,Rapid detection and differentiation of
wild-type and three attenuated lapinized vaccine strains of
Classical swine fever virus by reverse transcription polymerase
chain reaction, J Vet Diagn Invest, 2008, pp. 448-456, vol. 20.
cited by other.
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Primary Examiner: Benzion; Gary
Assistant Examiner: Wilder; Cynthia B
Attorney, Agent or Firm: WPAT, P.C. King; Anthony
Claims
What is claimed is:
1. A method for distinguishing wild-type classical swine fever
virus (CSFV) from lapinized CSF vaccine viruses in an infected
subject comprising: a) extracting total RNA from said subject; b)
providing a pair of primers for polymerase chain reaction (PCR)
comprising a forward degenerate primer selected from SEQ ID NO:3
and a reverse degenerate primer selected from SEQ ID NO:4, SEQ ID
NO:6 or SEQ ID NO:7; c) using said RNA as template and performing a
reverse transcription-PCR (RT-PCR) with said pair of primers
provided in step b); and d) determining the existence of said
wild-type CSFV or said lapinized CSF vaccine viruses according to
RT-PCR products which said vaccine viruses is at least 12
nucleotides longer than said wild-type CSFV by electrophoresis.
2. The method as claimed in claim 1, further comprising: providing
a pair of CSFV specific primers designed from the upstream and
downstream sequence of an at least 12 nucleotides insertion in the
3'-untranslated region of the lapinized CSF vaccine viruses as
compared to the wild-type CSFV; and using the pair of CSFV specific
primers from step b) with the product of the RT-PCR to perform a
nest-PCR.
3. The method as claimed in claim 1, further comprising a
comparison of a gel electrophoresis analysis result for a RT-PCR
product of a sample and said referenced lapinized CSF vaccine
viruses.
4. The method as claimed in claim 2, wherein said pair of CSFV
specific primers for RT-PCR comprises the following nucleotide
sequences: SEQ ID NO:5 and SEQ ID NO:6, and said pair of CSFV
specific primers for nest-PCR comprises: SEQ ID NO:3 and SEQ ID
NO:4.
5. The method as claimed in claim 2, wherein said pair of CSFV
specific primers for said RT-PCR comprises: SEQ ID NO:5 and SEQ ID
NO:6, and said pair of CSFV specific primers for nest-PCR
comprises: SEQ ID NO:3 and SEQ ID NO:7.
Description
FIELD OF THE INVENTION
This invention relates to a method and a diagnostic kit for
detection of the classical swine fever virus (CSFV), in particular
the differentiation field isolates and lapinized vaccine strains of
CSFV thereof, by reverse transcription-polymerase chain reaction
(RT-PCR), alone or in combination with nest-PCR (n-PCR), in which
at least a pair of CSFV-specific primers, each of which was
designed based on the conserved sequences within the
3'-untranslated region of CSFV's genome, are used.
BACKGROUND OF THE INVENTION
Classical swine fever (CSF), previously referred to hog cholera
(HC), is an important infectious disease of swine caused by CSFV.
Domestic pigs and wild boar are susceptible to CSFV. The virus
belongs to the genus Pestivirus within the family Flaviviridae,
which also includes bovine viral diarrhea virus (BVDV) and border
disease virus (BDV) (Matthaeus, Zbl. Vet. Med. 328: 126-132, 1981).
They are antigenically and structurally closely related but CSFV
can be distinguished from BVDV and BDV in serological and
nucleotide differences (Paton, J Comp pathol 112: 215-236, 1995).
The most commonly used in laboratory CSF diagnostic methods, such
as immuno-fluorescence stain, enzyme linked immunosorbent assay
(ELISA) and RT-PCR, can hardly distinguish the field isolates of
CSFV from the lapinized CSF vaccine viruses. Records of CSF in
Taiwan date back to 1938 (Lee, Scientific Agri (Taiwan), 2(11):
4-14). In an effort to control this highly contagious disease, a
live attenuated vaccines made from a lapinized CSF vaccine virus,
LPC strain, have been widely used in the field since 1958 in Taiwan
(Lin, National Science Council Special Publication Number 5, 1-42,
1981). Vaccination significantly decreased the incidence. However,
sporadic outbreaks were still reported occasionally. According to
the current legislation on CSF in Taiwan, each piglet has to be
vaccinated twice with the LPC vaccine virus in 3, 6 or 6, 9 weeks
old, it depend on the decrease level of maternal antibody.
Unfortunately, the vaccine virus can be detected and can't be
differentiated from field isolates of CSFV by ELISA and RT-PCR in
samples of pigs. Since the LPC vaccine virus interfered with the
laboratory diagnosis of CSF, the RT-PCR amplicons always proceed
with nucleotide sequencing to exclude the interference by LPC
vaccine virus in Taiwan.
A tentative assignment of world isolates of CSFV by genotyping has
been divided it into three groups with three or four subgroups:
1.1, 1.2, 1.3; 2.1, 2.2, 2.3; 3.1, 3.2, 3.3, 3.4 (Paton, Vet.
Microbiol. 73: 137-157, 2000). Phylogenetic analysis of the
E.sup.rns and E2 sequences of 158 CSFVs, which were isolated in the
field in Taiwan between 1989 and 2003, shows that four distinct
CSFVs genotypes existed in Taiwan including one endemic strain
(subgroup 3.4) and three introduced strains (subgroup 2.1a, 2.1b
and 2.2). The analysis also shows the LPC strain doesn't belong to
the aforementioned four subgroups, but to the subgroup 1.1. (Pan,
Arch Virol, 150(6): 1101-19, 2005).
There are four lapinized CSF vaccine viruses, namely LPC, HCLV,
Chinese C and Riem C, widely utilized in the world nowadays. The
LPC strain was derived from the Rova strain of CSFV, which had
already undergone about 250 serial passages in rabbits by Lederle
Laboratory in Philippines and was introduced into Taiwan by Dr.
Chung-Tao LEE in 1952 (Lee, Scientific Agri (Taiwan), 2(11): 4-14).
The pigs inoculated with this virus showed a severe post-vaccinated
reaction and a few of them even died of CSF after vaccination. In
order to obtain a highly safe and potent strain for CSF
vaccination, the virus was then rapidly and carefully
serial-passaged through native Taiwan rabbits. After more than 800
passages in rabbits, it proved extremely safe for pigs and highly
effective against CSFV (Lin, National Science Council Special
Publication Number 5, 1-42, 1981). Nowadays, the LPC vaccine is
widely utilized to protect pigs from CSF in Taiwan. The HCLV strain
was derived from the wildtype strain Shimen by 480 passages in the
bodies of rabbits in China in 1950s. (Wu, Virus Genes, 23(1):
69-76, 2001). The Chinese C strain is a cell culture adapted
derivative of HCLV strain. (Oleksiewicz, Veterinary microbiology,
92:311-325, 2003). The Riem C strain is a cell culture adapted
derivative of HCLV strain and used as bait vaccine in Europe
(Oleksiewicz, Veterinary microbiology; 92:311-325, 2003). Wu et al.
(Virus Genes, 23(1): 69-76, 2001) have sequenced HCLV strain and
discovered one notable insertion of 12 continuous nucleotides,
CTTTTTTCTTTT (SEQ ID NO:8) in the 3'-untranslated region of HCLV
genomic cDNA when compared with its parental virulent Shimen
strain. Wong et al (Virus Genes; 23(2): 187-92, 2001) also
sequenced the whole genome of LPC vaccine strain and found that an
insertion 0113 nucleotides, TTT(C/T)CTTTTTTTT SEQ ID NO:9, in the
3-untranslated region of LPC vaccine strain. The inventors of the
present invention had also compared all the CSFV sequences from the
GenBank and found that only the four lapinized CSF vaccine viruses,
LPC, HCLV, Chinese C and Riem C strains, have an insertion of
12.about.13 nucleotides in their 3-untranslated regions and the
insertion is not found in the field isolates of CSFV. Other
non-lapinized CSF vaccine viruses, such as Japanese GPE- and
Russian CS vaccine strains, also do not have the insertion.
Vaccination is one of the most successful tools for the prevention
of CSFV infection. Unfortunately, the use of laboratory diagnostic
methods to detect CSFV could be interfered by vaccine virus when
attenuated vaccines are in use. For this reason it is of interest
to know how long after vaccination can the vaccine strain be
detected in samples that are commonly used for diagnostic
procedures. To study the duration of vaccine virus distribution in
piglets, Lorena et al. (Veterinary microbiology, 81: 1-8, 2001)
inoculated piglets with the Chinese C strain vaccine virus and
studied the distribution of vaccine virus in organ samples of
inoculated piglets. He found that the virus can be detected in
tonsil on post-inoculation day (PID) 6, 8, 10, 13 and 16 using
ELISA and in blood samples on PID 2, 4, 6, 8, 10, 13, and 16 using
RT-PCR. Therefore, he emphasized that this factor must be
considered in routine diagnostic procedure, when vaccination
against CSF with a live vaccine is carried out. In Germany, CSF was
present in wild boar in different federal states (Veterinary
microbiology, 82: 301-310, 2001). Infection in domestic pigs was
usually caused by direct or indirect contacts with infected wild
boars. Wild boars distributed in the woods and they are difficult
to be caught for injecting CSF vaccine. Therefore, Oral application
of CSF vaccines (lapinised or cell culture vaccines) is necessary
and has been investigated in Europe (Veterinary microbiology, 73:
239-252, 2000). Kaden et al. (J Vet Med B Infect Dis Vet Public
Health, 51(6): 260-2, 2004) studied the persistence period of the
Chinese C strain vaccine virus in immunized animals after oral
vaccination. The results show that the C strain virus can be found
in organs until day 8 post-vaccination (pv) in domestic pigs and
until day 9 pv in wild boars. In the CSF endemic countries like
Taiwan where vaccination program with live vaccine is carried out,
the vaccine virus can probably be detected in the blood and
lymphatic tissue samples such as tonsil, lymph nodes and spleen.
Therefore, diagnosis with the commonly used diagnostic methods such
as immuno-fluorescence stain, ELISA and RT-PCR can be interfered by
the vaccine virus.
Virus isolation, ELISA and RT-PCR are the most commonly used
methods for CSF laboratory diagnosis. Paton et al. (Veterinary
microbiology 73: 159-174, 2000) show that the order of the
sensitivity was RT-nested PCR>RT-PCR>virus isolation>ELISA
when applying these methods to clinical samples in CSF diagnosis.
Dewulf et al. (Journal of Virological Methods 119: 137-143, 2004)
compare several CSF laboratory diagnostic techniques on live
animals for detection of infection. He concluded that the RT-nPCR
technique is the best diagnostic tool available for early detection
of a CSF infection. A real-time RT-PCR for the simple and rapid
diagnosis of CSF has been developed and evaluated in experimentally
infected swine and clinical samples (Risatti, Journal of Clinical
Microbiology; 41(1): 500-505, 2003; Risatti, Journal of Clinical
Microbiology, 43(1): 468-471, 2005). Accordingly, real-time RT-PCR
is recognized as a sensitive method for rapid diagnosis of CSF.
However, no real-time RT-PCR for distinguishing the field isolates
of CSFV from the lapinized CSF vaccine viruses has been
established.
The RT-PCR and nucleotide sequencing are widely used as the methods
to solve this problem. The disadvantage of these methods includes
the laborious process of the methods and the incapability of
screening large field samples. Zaberezhny et al. (Dtsch Tierarztl
Wochenschr. September; 106(9): 394-7, 1999) have used RT-PCR and
restriction enzyme digestion for differentiation between Russian
vaccine strain from field isolates of CSFV. Vilcek et al. (Acta Vet
Scand. 39(3): 395-400, 1998) also used restriction endonuclease
cleavage of PCR amplicons to distinguish the vaccine strain from
European field strains. These are the two documents available about
the utilization of the RT-PCR and restriction enzyme digestion
method to differentiate the vaccine virus and field isolates of
CSFV; besides, the prior art can only distinguish the two viruses
by RT-PCR followed by nucleotide sequencing. Thus there is no prior
disclosed information concerning the utilization of the
characteristic of one 12.about.13 nucleotides inserting in the
genome of the 3'-untranslated region of the lapinized CSF vaccine
viruses to establish a differential RT-PCR without combination with
other technique. CSFV specific primers are designed to amplify the
aforementioned 3'-untranslated region and then the size of the
RT-PCR amplicons can be compared directly by electrophoresis
without further processing the complicated enzymatic digestion and
nucleotide sequencing to determine the existence of the field
isolates of CSFV and lapinized CSF vaccine viruses and to
differentiate between them. The diagnosis of CSF can thus be more
rapid, convenient and the interference from the vaccine virus can
be more correctly excluded.
SUMMARY OF THE INVENTION
For solving the problem of the interference from the lapinized CSF
vaccine viruses with the laboratory diagnosis of CSF. This
invention relates to a method and a diagnostic kit for detection of
CSFV and differentiation of field isolates and lapinized vaccine
strains, by RT-PCR, alone or in combination with nest-PCR. By a
pair (or pairs) of CSFV specific primers designed from the
conserved region of the 3'-untranslated region of CSFV which there
are an insertion of 12.about.13 nucleotides in the 3'-untranslated
region of the lapinized CSF vaccine viruses, in comparison with the
corresponding region of field isolates of CSFV, to perform the
nucleotide expansion methods, such as RT-PCR or n-PCR, and to
separate the PCR products by electrophoresis (the better embodiment
is by using the 3-4% agarose gels) for direct analysis of the
results. The size of the RT-PCR amplicons can be compared directly
by electrophoresis without further processing the complicated
enzymatic digestion and nucleotide sequencing to determine the
existence of the field isolates and lapinized CSF vaccine virus and
to differentiate between them. Therefore, the field isolates of
CSFV can be rapidly differentiating from lapinized CSF vaccine
viruses.
The term "field isolates of CSFV" used in the present invention
refers generally to the CSFV isolated from the pigs in the field
and not serial-passaged in rabbits or other animals.
The term "lapinized classical swine fever vaccine viruses" used in
the present invention refers to the CSFV strain obtained from high
passages in rabbits for further utilization as CSF vaccine. Four
attenuated lapinized CSF vaccine viruses nowadays widely utilized
in the world as a vaccine: the LPC strain, the HCLV strain, the
Chinese C strain and the Riem strain.
The term "nucleic acid" used in the present invention refers to DNA
(deoxyribonucleic acid) or RNA (ribonucleic acid) and their single
or double stranded polymers (poly nucleotides). Unless limited, the
term includes those nucleic acids having similar binding
characteristics with the reference nucleic acid and those natural
nucleic acid analogues already known and metabolize in a similar
way as natural nucleic acids.
The term "same" or "unique" in percentage about two or more
nucleotide or protein sequences refers to two or more sequences or
sub-sequences which have the same or the same percentage of the
amino acid residues or nucleotides, while using one of the
sequence-comparison algorithms (e.g. Smith-Waterman algorithm) or
processing the maximum-corresponding comparison and ranking by
visual measure mentioned in this text.
Concerning the sequence-comparison, a section of sequence is taken
as a reference for the testing sequence to compare. When applying
sequence-comparison algorithm, the testing sequence is entered into
the computer as well as the reference one, the sub-sequence
coordinate can be assigned if necessary, and the parameters of the
sequence algorithm program should be set. The sequence-comparison
algorithm can then, based on the program parameters set, calculate
the percentage of uniqueness of the testing sequence compared to
the reference sequence.
The term "amplification of DNA or RNA sequence" used in the present
invention refers to all methods that allow the amplification of the
number of the sample of the targeted nucleotide sequence. The most
widely used nucleotide sequence expansion technology is PCR.
Basically, it means attaching two primers, which are respectively
sense primer and antisense primer to the denaturalized DNA stroma
and extending by using thermo-stable DNA polymerase. The later can
catalyze RT-PCR (reverse-transcript PCR) and real-time PCR in rapid
and repeating cycles. (In a three-step PCR, it means
denaturalization/annealing/extension; in a two-step PCR, it means
denaturalization/annealing and extension.). RT-PCR can be performed
with a single thermo-stable enzyme with reverse-transcription
enzymes and DNA polymerase (Maire and Jefferan, 1999). Optionally,
it can also be performed with a single test-tube reaction (Kathy et
al., biological technology 17:1034-1036, 1994) containing both
enzymes (reverse-transcription enzyme and thermo-stable DNA
polymerase).
The term "nest polymerase chain reaction (nest-PCR or n-PCR)"
refers to the improved design which can increase the sensitive of
PCR, i.e. the secondary PCR which concerns the design of a pair of
primers on the PCR product to perform the secondary PCR, the
sensitivity can thus increase by 100 to 1000 times.
The term "3'-untranslated region of CSFV nucleic acid" is defined
as the 3'-untranslated region of nucleotide-sequence of the LPC
vaccine virus (SEQ ID NO: 1) which corresponds to the nucleotide
section (SEQ ID NO: 2) from 11,874 to 12,240 base pairs (bp) of 3'
nucleotide positioned on Alfort 187 strain (Accession No: X87939)
of the CSFV and includes other isogenic classical swine fever
vaccine strains attenuated by rabbits such as the 3' nucleotide
sequence of LPC, HCLV, Chinese C and Riem strain. According to the
present invention, the 3'-untranslated region of the nucleotide of
lapinized CSF vaccine strains comprises an insertion of 12.about.13
nucleotides in comparison with the field isolates of CSFV
(equivalent to the poly T segment "CTTTTTTTCTTTT" as shown in SEQ
ID NO: 1).
The term "comprising equivalent nucleotide as assigned SEQ ID NO"
refers that the nucleotide region defined by the SEQ ID NO (or any
equivalence or any related part) is included in the interested
nucleotide molecule. The term "equivalence" in the present
invention includes variants (such as polymorphic variants) and
derivatives (such as modified variants).
The term "primer" refers to a short nucleic acid structure, which
can be the starting point of the replication of nucleic acid. While
carrying out the PCR reaction, a pair of primers is need, one
positive-strand and one negative-strand situated respectively at
the both end. The region defined by this pair of primers is the
size of the PCR product. The oligonucleotide used in the PCR
reaction for expanding the desired nucleic acid sequence can be
double-strand or single-strand. In the present invention, the
primer can be synthesized (e.g. chemically synthesized), for
example, by the habitually know chemical method of
phosphate-triester or phosphite amide. Primers can also be produced
in vitro by the nucleotide sequence expansion method. If the
expanded oligonucleotide is double-strand, then it can be converted
to a single-strand molecule protect the single-strand
oligonucleotide from the active action of the exonuclease.
Furthermore, the primers mentioned in this invention are derived
from the recombination plasmid of the insert which has the
corresponding nucleotide sequence; the later can be split and
retrieved from the selected plasmid by using the proper nucleotide
if needed, splitting method such as classification of molecular
weight can be used.
According to the method of the present invention, a conserved
sequence within 3'-untranslated nucleotide sequence (SEQ ID NO:1)
equivalent to LPC is chosen to design a pair of CSFV specific
primers for performing RT-PCR reaction to distinguish the field
isolates of CSFV from the lapinized vaccine virus by comparing to
the size of the segment on the electrophoresis film. As for those
who are familiar with the prior art, the letter "R" in the
nucleotide sequence of the primers mentioned in this text refers to
the base A or G unless otherwise specified.
In the preferred embodiment of this invention, the specific primers
of CP-3F (5'-ACCCTRTTGTARATAACACTA-3'), identified as SEQ ID NO:3,
and CP-3R (5'-GTTAAAAATGAGTGTAGTGTGGTA-3'), identified as SEQ ID
NO:4 designed from the conserved region are used to amplify the
different genotypes of CSFV to perform RT-PCR amplification. The
length of the RT-PCR product after amplification is 127 bp for
field isolates of CSFV and 140 bp for LPC vaccine virus. Therefore,
the field isolates of CSFV and the lapinized vaccine strain can
directly be distinguished from the result of the electrophoresis
analysis without performing the DNA sequencing. In another
embodiment of this invention, for increasing the sensitivity of the
test, a nest-PCR (n-PCR) CSFV testing method is designed as to
first amplify the CSFV specific primers CP-5
(5'-GTAGCAAGACTGGAAATAGGTA-3'), identified as SEQ ID NO:5, and CP-6
(5'-AAAGTGCTGTTAAAAATGAGTG-3'), identified as SEQ ID NO:6, of
different genotypes of CSFV for the first RT-PCR amplification,
then the product of the first amplification (the length for field
isolates of CSFV is 367 bp and LPC vaccine virus is 380 bp
respectively); is taken as template to perform nest-PCR using CP-3F
and CP-3R as primers. The length of the obtained PCR product is 127
bp for field isolates of CSFV and 140 bp for LPC vaccine virus. In
one other embodiment, using CP-3F (5'-ACCCTRTTGTARATAACACTA-3'),
identified as SEQ ID NO:3, and CP-9R
(5'-GTACCAGTTCTTRCTCATTCAATA-3'), identified as SEQ ID NO:7, as
primers, the amplified product of nest-PCR is 89 bp for field
isolates of CSFV and 102 bp for LPC vaccine virus; only that the
PCR product using this pair of primers is less. The nest-PCR is of
high sensitivity and can conduct a high-quality testing result to
chronic classical swine fever with low virus titer or CSFV carrier
pigs with normal appearance.
According to the method of this invention, a RT-PCR diagnostic kit
for determining the existence of field isolates of CSFV or
lapinized vaccine virus in the samples is developed, either by a
one-step RT-PCR diagnostic method to detect the CSFV or/and by
furthermore using the nest-PCR to distinguish the filed isolates of
CSFV from lapinized CSF vaccine viruses.
The term "samples" in this text refers to any biological material
directly collected from a seemingly infected swine or enriched
ones. The biological material can be any expectoration, bronchus
alveolar lavage, blood, skin tissue, lymphatic tissue, living
tissue specimens, semen, cultivated lymphocyte or blood, specimen
of excrement and urine. The biological material can also be culture
object or solution of artificially infected cellules.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the electrophoretogram showing the result of
electrophoresis (with a 4% agarose gel) of the product of the
one-step RT-PCR (using CP-3F and CP-3R as primers) of the field
isolates of CSFV and lapinized CSF vaccine virus from different
sources. Wherein Lane 1 is 100 bp Ladder marker, Lane 2 is the
92-TC1 strain of field isolate of CSFV (2.1a subgroup), Lane 3 is
the 90-YL5 strain of field isolate of CSFV (2.1b subgroup), Lane 4
is the 84-KS1 strain of field isolate of CSFV (2.2 subgroup), Lane
5 is the S-59 strain of field isolate of CSFV (3.4 subgroup), Lane
6 is the 93-TD1 strain, which is a field isolate of LPC vaccine
virus (1.1 subgroup), Lane 7 is the positive control of the LPC
vaccine virus, Lane 8 is ALD strain as the positive control, which
is a virulent CSFV, Lane 9 is the BVDV, Lane 10 is the negative
control and Lane 11 is the 100 bp Ladder marker.
FIG. 2 is the electrophoretogram showing the result of
electrophoresis (with a 2% agarose gel) of the product of the
one-step RT-PCR (using CP-5 and CP-6 as primers) of the field
isolate of CSFV and lapinized CSF vaccine virus from different
sources. Wherein Lane 1 is the 100 bp Ladder marker, Lane 2 is the
92-TC1 strain of field isolate of CSFV (2.1a subgroup), Lane 3 is
the 90-YL5 strain of field isolate of CSFV (2.1b subgroup), Lane 4
is the 84-KS1 strain of field isolate of CSFV (2.2 subgroup), Lane
5 is the S-59 strain of field isolate of CSFV (3.4 subgroup), Lane
6 is the 93-TD1 strain, which is a field isolate of LPC vaccine
virus (1.1 subgroup), Lane 7 is the positive control of the LPC
vaccine strain, Lane 8 is ALD strain as the positive control, which
is a virulent CSFV, Lane 9 is the BVDV, Lane 10 is the negative
control and Lane 11 is the 100 bp Ladder marker.
FIG. 3 is the electrophoretogram showing the result of
electrophoresis (with a 4% agarose gel) of the product of the
nest-PCR (using CP-3F and CP-3R as primers) of the field isolate of
CSFV and lapinized CSF vaccine virus from different sources.
Wherein Lane 1 is the 100 bp Ladder marker, Lane 2 is the 92-TC1
strain of field isolate of CSFV (2.1a subgroup), Lane 3 is the
90-YL5 strain of field isolate of CSFV (2.1b subgroup), Lane 4 is
the 84-KS1 strain of field isolate of CSFV (2.2 subgroup), Lane 5
is the S-59 strain of field isolate of CSFV (3.4 subgroup), Lane 6
is the 93-TD1 strain, which is a field isolate of LPC vaccine virus
(1.1 subgroup), Lane 7 is the positive control of the LPC vaccine
virus, Lane 8 is ALD strain as the positive control, which is a
virulent CSFV, Lane 9 is the BVDV, Lane 10 is the negative control
and Lane 11 is the 100 bp Ladder marker.
FIG. 4 shows SEQ ID NO: 1. The sequence of LPC vaccine virus
between primers CP5 and CP6 (length 380 bp).
FIG. 5 shows SEQ ID NO: 2. The sequence of Alfort187 strain of
CSFV, between primers CP5 and CP6 (length 367 bp), sequence
position on the Alfort187 (Accession No: X87939) is from 11,874 to
12,240.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Although the present invention is to be explained in relation to
its preferred embodiment as illustrated in the following examples,
it is not meant to limit the scope of the invention. It is to be
understood that many other possible modifications and variations
can be made by those skilled in the art without departing from the
spirit and scope of the invention claimed.
Example 1
A one-step RT-PCR for distinguishing the field isolates of CSFV
from the lapinized CSF vaccine viruses.
(1) Design of the Primers
A pair of amplification primers is designed from the conserved
sequence within 3'-untranslated region of CSFV. This primer is a
CSFV specific primer, which can only amplify the CFS virus but not
its two close relatives bovine viral diarrhea virus (BVDV) and
border disease virus (BDV). This primer is also one of the
universal primers, which can be used to amplify different genotypes
of CSFV. The nucleotide sequence of this primer pair is as
follows:
TABLE-US-00001 (SEQ ID NO:3) CP-3F (5'-ACCCTRTTGTARATAACACTA-3')
(SEQ ID NO:4) CP-3R (5'-GTTAAAAATGAGTGTAGTGTGGTA-3')
(2) Virus Sources
The lymphatic tissues of internal organs of swine such as tonsil,
lymph node and spleen are ground with a mortar, then MEM medium
(minimal essential medium) is added to form a 10% (w/v) suspension.
After 20 minutes of 3000.times.g centrifugation, the top layer is
retrieved for performing the RT-PCR. Alternatively, the cell
culture of PK-15 cell infected by CSFV can also be used to perform
RT-PCR.
(3) Extraction of Nucleic Acid
Add 100 .mu.L of the top layer of the above-mentioned swine tissue
emulsion or the cell culture solution into 1 mL of TRIzol total RNA
extraction reagent. Mix with a vibrator for 30 seconds, place under
room temperature for 5 minutes, add in 200 .mu.L of chloroform,
after 15 seconds of mixing, leave it still for another 3 minutes,
centrifuge at 4.degree. C. and 12,000 rpm for 15 minutes, retrieve
the top layer and put in a clean centrifugal tube, add an
equivalent volume of isopropyl alcohol and shake it up and down to
get a uniform mixture, leave it still under room temperature for 10
minutes, centrifuge at 4.degree. C. and 12,000 rpm for 15 minutes,
remove all the solution, leave it still under room temperature for
10 to 15 minutes, and add 100 .mu.L RNase-free secondary distilled
water to dissolve it for further use.
(4) Single-Tube RT-PCR
This experiment is a single-tube one-step reaction, i.e. the
reverse transcription (RT) and the polymerase chain reaction (PCR)
proceed in a single reaction tube. All the necessary reagents are
added into the same reaction tube at the same time. The
characteristic of this experiment is the easy operation, which
saves time and labor. Take 5 .mu.L of the prepared nucleic acid
sample, add the pair of CSFV specific primers CP-3F and CP-3R (20
pm), 5 .mu.L of 10.times. Super Thermal buffer, 2 Unite (U) of
RNase inhibitor (Promega), 2 U of AMV reverse transcriptase
(Promega), 1 U of polymerase and 8 .mu.L (1.25 mM) of dNTP,
furthermore, add DEPC treated secondary distilled water to bring
the final reaction volume to 50 .mu.L. In the thermal circulation
reaction machine (ABI 9600), the reverse transcription reaction is
carried out at 42.degree. C. for 40 minutes, then the PCR reaction
is carried out at 95.degree. C. for 1 minute, followed by 35
consecutive rounds of 94.degree. C. for 40 seconds, 55.degree. C.
for 40 seconds, and 72.degree. C. for 40 seconds, then the tube is
kept at 72.degree. C. for 7 minutes and stored at 4.degree. C. The
product of the RT-PCR reaction is analyzed by electrophoresis with
4% of agarose gel. As shown in the electrophoretogram of FIG. 1,
the length of the products of one-step RT-PCR is 127 bp for field
isolates of CSFV and 140 bp for LPC vaccine virus,
respectively.
Example 2
For increasing the sensitivity of the test, another method using
nest-PCR is designed for distinguishing the field isolates of CSFV
from the lapinized CSF vaccine virus in addition to the one-step
RT-PCR to detect the CSFV. The method is as follows:
(1) Design of the Primers
A pair of amplification primers is designed from the outer side of
the amplification region of the CP-3F and CP-3R primers. This pair
of primers is CSFV specific primers, which only amplifies the CSFV
but not the two close relatives, BVDV and BDV. This pair of primers
is one of the universal primers designed from the conserved
sequences of CSFV, i.e. it can be used for CSF virus of all
genotypes. The nucleotide sequence of this pair of primers is as
follows:
TABLE-US-00002 (SEQ ID NO:5) CP-5 5'-GTAGCAAGACTGGAAATAGGTA-3' (SEQ
ID NO:6) CP-6 5'-AAAGTGCTGTTAAAAATGAGTG-3'
(2) Single-Tube RT-PCR
Take 5 .mu.L of the prepared nucleic acid sample, add the pair of
CSFV specific primers CP-5 and CP-6 (20 pm), 5 .mu.L of 10.times.
Super Thermal buffer, 2 U of RNase inhibitor (Promega), 2 U of AMV
reverse transcriptase (Promega), 1 U of polymerase and 8 .mu.L
(1.25 mM) of dNTP, furthermore, add DEPC treated secondary
distilled water to bring the final reaction volume to 50 .mu.L. In
the thermal circulation reaction machine (ABI 9600), the RT
reaction is carried out at 42.degree. C. for 40 minutes, then the
PCR reaction is carried out at 94.degree. C. for 1 minute, followed
by 35 consecutive rounds of 94.degree. C. for 40 seconds,
55.degree. C. for 40 seconds, and 72.degree. C. for 40 seconds,
then the tube is kept at 72.degree. C. for 7 minutes and stored at
4.degree. C. The product of the RT-PCR reaction is analyzed by
electrophoresis with 2% of agarose gel. As shown in the
electrophoretogram of FIG. 2, the length of one-step RT-PCR
products is 367 bp for field isolates of CSFV and 380 bp for LPC
vaccine virus, respectively.
(3) Nest-PCR for Distinguishing the Field Isolates of CSFV From the
Lapinized CSF Vaccine Virus
2 .mu.L of the first PCR product is taken as template. Add the
above-mentioned primers CP-3F and CP-3R (20 pm), 5 .mu.L of
10.times. Super Thermal buffer, 1 U of DNA polymerase and 8 .mu.L
(1.25 mM) of dNTP, furthermore, add DEPC treated secondary
distilled water to bring the final reaction volume to 50 .mu.L. In
the thermal circulation reaction machine (ABI 9600), the polymerase
chain reaction is carried out directly under 35 consecutive rounds
of 94.degree. C. for 35 seconds, 55.degree. C. for 30 seconds, and
72.degree. for 30 seconds, then the tube is kept at 72.degree. C.
for 7 minutes and stored at 4.degree. C. The RT-PCR reaction
solution is then subjected to electrophoresis for 40 minutes with
agarophyte gel solution. As shown in the electrophoretogram of FIG.
3, the length of the products of nest-PCR is 127 bp for field
isolates of CSFV and 140 bp for LPC vaccine virus,
respectively.
As can been seen from the experimental results above, this
invention provides a rapid and easy method to distinguish and
identify the field isolates of CSFV and lapinized CSF vaccine virus
based on the electrophoresis result without further performing
nucleotide-sequencing or enzymetic digestion.
While the present invention has been described above in terms of
specific embodiments, it is to be understood that the invention is
not limited to these disclosed embodiments. Many modifications and
other embodiments of the invention will come to mind of those
skilled in the art to which this invention pertains; they are
intended to be and are covered by both this disclosure and the
appended claims. It is intended that the scope of the invention be
determined by proper interpretation and construction of the
appended claims and their legal equivalents, as understood by those
skilled in the art relying upon the disclosure in this
specification and the attached drawings.
SEQUENCE LISTINGS
1
91380DNAArtificial SequenceLPC Vaccine 1gtagcaagac tggaaatagg
tacatgcccg gagaaggcca caccctgcaa ggaagacatt 60atgaagaatt ggtgttggca
aggaaacaga tcaacaactt tcaagggaca gacaggtaca 120atctaggccc
aatagtcaac atggtgttga ggaggctgag agtcttgatg atgaccttga
180tagggagagg ggtatgagcg cgggtaatct gggatctgaa cccgccagta
ggaccctatt 240gtagataaca ctaattttct ttttttcttt tttatttatt
tagattttat tatttattta 300tttatttatt tattgaatga gtaagaattg
gtacaaacta cctcaagtta ccacactaca 360ctcattttta acagcacttt
3802367DNAClassic Swine Fever Virusmisc_difference(1)..(367)
2gtagcaagac tggaaacagg tacatacctg gagagggcca caccctgcaa gggagacatt
60atgaagaact ggtgttggca agaaaacaga tcaataactt tcaagggaca gacaggtaca
120atctaggccc aatagtcaac atggtgttaa ggaggctgag agtcatgatg
atgaccctga 180tagggagagg ggtatgaacg cgggcaaccc gggatctgga
cccgccagta ggaccctatt 240gtagataaca ctaatttttt atttatttag
atattattat ttatttattt atttatttat 300tgaatgagta agaactggta
caaactacct caagttacca cactacactc atttttaaca 360gcacttt
367321DNAArtificial SequenceCP-3F primer 3accctrttgt arataacact a
21424DNAArtificial SequenceCP-3R primer 4gttaaaaatg agtgtagtgt ggta
24522DNAArtificial SequenceCP-5 primer 5gtagcaagac tggaaatagg ta
22622DNAArtificial SequenceCP-6 primer 6aaagtgctgt taaaaatgag tg
22724DNAArtificial SequenceCP-9R primer 7gtaccagttc ttrctcattc aata
24812DNAArtificial Sequence3'-untranslated region of HCLV genomic
cDNA 8cttttttctt tt 12913DNAArtificial Sequence3'-untranslated
region of LPC vaccine strain 9tttycttttt ttt 13
* * * * *